Ontogeny of electrically excitable cells in cultured olfactory epithelium.

Abstract

A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuronspecific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP. Immunohistochemistry. Rabbit anti-glial fibrillary acidic protein (GFAP) was obtained from A. Bignami, mouse monoclonal anti-rat T200 protein was obtained from R. Hyman, and monoclonal anti-S100 and anti-neuron-specific enolase was from B. Boss. For staining with anti-T200, cultures were incubated with the primary antibody for 30 min at room temperature. The cultures were then washed and exposed for an additional 30 min to a rhodamine-labeled goat antibody against mouse immunoglobulin (Tago, Burlingame, CA). Finally, the cultures were washed, air dried, and fixed with 95% ethanol for 10 min at -20°C. For labeling with anti-GFAP, cultures were first fixed with 95% ethanol for 10 min at -20°C, dried, and then incubated for 30 min with the primary antiserum. After washing, the cultures were exposed to a fluorescein-labeled goat antibody against rabbit immunoglobulin for an additional 30 min, followed by more washing and a final air drying. For double-labeling the cells were first stained with anti-T200, fixed, and then stained with anti-GFAP. Staining with anti-S100 and anti-nerve-specific enolase was done according to published procedures (8).

Cell migration from the transplanted olfactory placode in Xenopus

“…Several previous studies support the view that the olfactory placode can generate a variety of neurons and sheath cells (Doucette 1989;Farbman and Squinto Mendoza et al 1982;Murakami et al 1991Murakami et al , 1992Schubert et al 1985;Schwanzel-Fukuda and Pfaff 1989;Wray et al 1989a, b). It is possible that cells of placodal origin incorporated into the brain of the host under our experimental conditions are induced to differentiate and to express a different phenotype spectrum from the one typically expressed by cells that mature in their normal habitat.…”

“…It has been suggested that some cells migrating along the fascicles of the olfactory nerves could be incorporated into the olfactory bulb as a special type of glial cell (Doucette 1989). According to recent in vitro studies, cells derived from the olfactory epithelium could act as pluripotent precursors for both glial and neuronal cells (Schubert et al 1985). Moreover, recent transplantation studies have demonstrated that cells isolated from the embryonic and postnatal epithelia of the rat, when transplanted into the CNS of embryonic rats at E15, can change their fate and become either glia or neurons according to their new final location (Magrassi and Graziadei 1994).…”

Cell cultures of neuroblasts from rat olfactory epithelium that show odorant responses.

“…Biochemical studies (3)(4)(5) clearly have demonstrated an odorant-dependent increase of adenylyl cyclase activity in preparations of cilia from frog or rat OE. It seemed that the SNIF cell strains, derived from the OE, might be capable of responding to odorant stimuli by production of a second messenger such as cAMP.…”

“…Past attempts to grow these cells in culture (3,10,11) did not yield strains that were differentiated sufficiently to suggest their use in the study of olfaction in vitro. We report here that two of three cloned cell strains, although they appear to be incompletely differentiated neurons in culture, have nevertheless responded sensitively and selectively to test odorants by accumulating cAMP.…”

“…In the OE these cells are surrounded by sustentacular cells that are secretory and may also serve a glia-like function for the sensory neurons. At the base of this pseudostratified epithelium lie the basal cells, which divide and give rise throughout life to new sensory neurons (1,2) and possibly also to new sustentacular cells (1)(2)(3).…”